Vaccine

ABSTRACT

The present invention provides an optimal formulation of multiple serotype  Streptococcus pneumoniae  conjugate vaccines.

FIELD OF THE INVENTION

The present invention relates to an improved Streptococcus pneumoniavaccine.

BACKGROUND OF THE INVENTION

Children less than 2 years of age do not mount an immune response tomost polysaccharide vaccines, so it has been necessary to render thepolysaccharides immunogenic by chemical conjugation to a proteincarrier. Coupling the polysaccharide, a T-independent antigen, to aprotein, a T-dependent antigen, confers upon the polysaccharide theproperties of T dependency including isotype switching, affinitymaturation, and memory induction.

However, there can be issues with repeat administration ofpolysaccharide-protein conjugates, or the combination ofpolysaccharide-protein conjugates to form multivalent vaccines. Forexample, it has been reported that a Haemophilis influenzae type bpolysaccharide (PRP) vaccine using tetanus toxoid (TT) as the proteincarrier was tested in a dosage-range with simultaneous immunization with(free) TT and a pneumococcal polysaccharide-TT conjugate vaccinefollowing a standard infant schedule. As the dosage of the pneumococcalvaccine was increased, the immune response to the PRP polysaccharideportion of the Hib conjugate vaccine was decreased, indicating immuneinterference of the polysaccharide, possibly via the use of the samecarrier protein (Dagan et al., Infect Immun. (1998); 66: 2093-2098).

The effect of the carrier-protein dosage on the humoral response to theprotein itself has also proven to be multifaceted. In human infants asit was reported that increasing the dosage of a tetravalent tetanustoxoid conjugate resulted in a decreased response to the tetanus carrier(Dagan et al., supra). Classical analysis of these effects ofcombination vaccines have been described as carrier induced epitopicsuppression, which is not fully understood, but believed to result froman excess amount of carrier protein (Fattom, Vaccine 17: 126 (1999)).This appears to result in competition for Th-cells, by the B-cells tothe carrier protein, and B-cells to the polysaccharide. If the B-cellsto the carrier protein predominate, there are not enough Th-cellsavailable to provide the necessary help for the B-cells specific to thepolysaccharide. However, the observed immunological effects have beeninconsistent, with the total amount of carrier protein in some instancesincreasing the immune response, and in other cases diminishing theimmune response.

Hence there remain technical difficulties in combining multiplepolysaccharide conjugates into a single, efficacious, vaccineformulation. It is thus an object of the present invention to develop animproved formulation of a multiple serotype Streptococcus pneumoniaepolysaccharide conjugate vaccine.

SUMMARY OF THE INVENTION

In one aspect the present invention is an improved Streptococcuspneumonia vaccine comprising 11 or more polysaccharides from differentS. pneumonia serotypes conjugated to 2 or more carrier proteins, wherethe polysaccharides from serotypes 6B, 19F and 23F are conjugated to afirst carrier protein and the remaining serotypes are conjugated to 1 or2 secondary carrier proteins, and where the secondary carrier protein(s)are different from the first carrier protein. Preferably serotypes 6Band 23F are conjugated to the first carrier protein, and more preferablyonly serotype 6B is conjugated to the first carrier protein. In apreferred embodiment, one of the secondary carrier protein(s) is H.influenzae protein D. The present invention may further comprise S.pneumonia surface proteins, preferably from the PhtX family, the CbpXfamily, the CbpX truncate family and Ply.

In a related aspect, the present invention is an improved method toelicit a protective immune response to infants against S. pneumoniae byadministering the polysaccharide conjugate vaccine of the presentinvention.

In another related aspect, the present invention is an improved methodto elicit a protective immune response, that is, for the prevention oramelioration of pneumococcal infection in the elderly (e.g., pneumonia)and/or in infants (e.g., Otitis media), by administering thepolysaccharide conjugated vaccine of the present invention and a S.pneumonia surface protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the immune response to 12different pneumococcal polysaccharides as determined by the geometricmean fold increase after polysaccharide immunization.

FIG. 2 shows the geometric mean IgG concentration [GMC] (μg/ml) andOpsonic Titres on day 14 (Post II) after immunization of adult rats with1.0 μg PS-PD alone or combined in a tetravalent, pentavalent,heptavalent or decavalent vaccine.

FIG. 3 shows the GMC for 11 serotypes and PD (protein D) versus thedosage of 6B and 23F in one dimension, and the dosage of the 9 others inthe second dimension. The trend is always the same for all serotypes andPD. Increasing the dosage of 6B and 23F has a dramatic effect ondecreasing the immune response to tie remaining conjugates, even thoughthe dosage of those conjugates is unchanged.

FIG. 4 shows a graph of the IgG GMC in infant rats versus the totalamount of PD immunized for 11 serotypes. (i.e., by summing all the PDfrom each component at each dose). The general trend is that as thedosage of carrier protein increases, there is a decrease in the IgGresponse to all polysaccharides, and to PD itself. This overall trend isstrong evidence of carrier-induced epitopic suppression. However, thefact that the curve is not monotonous is an indication that there isanother factor involved which appears to depend on Serotype 6B.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an optimal formulation ofmultiple-serotype Streptococcus pneumoniae polysaccharide conjugatevaccines, by judicious selection of various polysaccharides conjugatedto different, or alternate, carrier proteins. The invention is based onthe fact that polysaccharide conjugates of one serotype may influence,or modulate, the immune response observed for other (serotype)polysaccharide conjugates. Thus, an optimal multi-valent polysaccharideconjugate vaccine can be prepared by putting different S. pneumoniapolysaccharides, with different immune regulatory properties, onalternative carrier proteins.

The present invention is based on the combination of several factors:(i) the dosage-response curve to polysaccharides is frequentlybell-shaped (Gaussian), with the maximal response at a dosagedistinctive for each polysaccharide (i.e., serotype or structure); (ii)the immunogenicity of certain polysaccharides is regulated with age inhumans and in animal models; (iii) the combination of S. pneumoniaepolysaccharide conjugates into multivalent formulations often results ina decrease in immunogenicity of one or more components of the vaccine;(iv) however, certain polysaccharide conjugates result in an enhancedimmune response when combined; (v) polysaccharides from serotypes 6B and23F, and to a lesser degree 19F can regulate the immune response ofother polysaccharides (i.e., other serotypes) when they are conjugatedto a common carrier protein.

Thus the present invention is based on the complex relationship of allthe above and, in contrast to prior studies, concludes that thebell-shaped dosage-response curve (i.e., which denotes peakimmunogenicity) of polysaccharide-protein conjugates is heavilyinfluenced by the quantity and nature of other polysaccharides. Thisimmunological effect is referred to as modulation. Moreover, it has beendiscovered that the modulation of polysaccharide conjugates occursthrough a common carrier protein. That is, a few polysaccharideconjugates may modulate the immune response to different polysaccharideconjugates, so long as they have a common carrier protein. Thus as notedabove, the invention is based on the judicious selectionpolysaccharides, to determine which polysaccharides should be conjugatedto the same or different carrier proteins.

As shown in more detail below: (a) certain S. pneumonia polysaccharides(PS), when conjugated, are strongly regulated with age, in particularserotypes 6B, 14, 19F and 23F. Serotypes 8, 12 and 18C are weaklyregulated with age. Serotypes 1, 2, 3, 4, 5, 7F and 9V are not regulatedwith age (see FIG. 1).

In addition (b), polysaccharides 1, 3, 6B, 9V and 23F, when combinedinto an 11-valent multiformulation, showed an increase in the immuneresponse elicited, as compared to a monovalent polysaccharide conjugate.In contrast, serotype 14 showed a significant decrease in themultivalent formulation (see FIG. 2).

Moreover (c), polysaccharides from serotypes 6B and 23F, and to a lesserdegree 19F can regulate the immune response of other polysaccharides(i.e., other serotypes) if they are conjugated to a common carrierprotein (see FIGS. 3 and 4).

Thus in one embodiment, the present invention comprises polysaccharides6B, 19F and 23F conjugated with one (a first) carrier protein, and theremaining polysaccharides are conjugated to an alternative (orsecondary) carrier protein(s), with the proviso that the primary andsecondary carrier proteins are different. Preferably, polysaccharides 6Band 23F are conjugated with the same carrier protein, and the remainingpolysaccharides are conjugated to a secondary carrier protein(s). Morepreferably, only polysaccharide 6B is conjugated to a primary (first)carrier protein and the remaining polysaccharides are conjugated to asecondary carrier protein(s).

The primary carrier protein need not be limited to a specificembodiment, but may include proteins or fragments thereof of DT(Diphtheria toxoid), TT (Tetanus toxoid), DT crm197 (a DT mutant), otherDT point mutants, (e.g.at position Glu-148, see, e.g., U.S. Pat. No.4,709,017, WO93/25210, WO95/33481), FragC (fragment of TT), Ply(pneumolysin and mutants thereof), PhtA, PhtB, PhtD, PhtE, (Pht A-E aredescribed in more detail below) OnipC (from N. meningitidis), PorB (fromN. meningitidis), etc. Preferably it is DT, TT or crm 197. Morepreferably it is DT.

The secondary carrier protein(s) will also be selected from the groupconsisting of PD (Haemophilus influenzae protein D—see, e.g., EP 0 594610 B), DT, TT, DT crm 197, FragC, Ply, PhtA, PhtB, PhtD, PhtE, OmpC,PorB, etc. It is contemplated that 2 different secondary carrierproteins may be used, but preferably, only one secondary carrier proteinis to be used in the present invention.

The number of S. pneumoniae polysaccharides can range from 11 differentserotypes (or “V”, valences) to 23 different serotypes (23V). Preferablyit is 11, 13 or 16 different serotypes. In another embodiment of theinvention, the vaccine may comprise conjugated S. pneumoniaepolysaccharides and unconjugated S. pneumoniae polysaccharides.Preferably, the total number of polysaccharide serotypes is less than orequal to 23. For example, the invention may comprise 11 conjugatedserotypes and 12 unconjugated polysaccharides. In a similar manner, thevaccine may comprise 13 or 16 conjugated polysaccharides and 10, or 7respectively, unconjugated polysaccharides.

Preferably the multivalent pneumococcal vaccine of the invention will beselected from the following serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V,10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F,although it is appreciated that one or two other serotypes could besubstituted depending on the age of the recipient receiving the vaccineand the geographical location where the vaccine will be administered.For example, an 11-valent vaccine may comprise polysaccharides fromserotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. A 13-valentpediatric (infant) vaccine may also include serotypes 6A and 19A,whereas a 13-valent elderly vaccine may include serotypes 8 and 12F.

Preferably, the Streptococcus polysaccharides of the invention aredepolymerized (sized) to a final range of 100-500 kD. Thus, anotherfeature of the present invention is the ratio of carrier protein topolysaccharide. For the conjugated polysaccharides, the ratio of carrierprotein to polysaccharide (P/PS) will be greater than 0.5 (i.e., >0.5,and up to 1.7) (w/w) for at least seven serotypes. Preferably the ratiois □0.70 to 1.5 (e.g., for at least serotypes 6B, 19F, 23F). Morepreferably the range will be 0.8 to 1.5 (e.g., for at least serotypes6B, 19F, 23F). Most preferably still, the ratio of P/PS will at leastapproach 1 (e.g., 0.9-1.1) for one or more serotypes of the invention(e.g., 4).

A related feature of the present invention is that the level ofunconjugated (free) carrier protein is less than 10% of the total amountof carrier protein, and that the level of unconjugated polysaccharide isless than 10% of the total amount of polysaccharide, for each serotype.

The polysaccharides may be linked to the carrier protein(s) by any knownmethod (for example, by Likhite, U.S. Pat. No. 4,372,945 by Armor etal., U.S. Pat. No. 4,474,757, and Jennings et al., U.S. Pat. No.4,356,170). Preferably, CDAP conjugation chemistry is carried out (seeWO95/08348).

In CDAP, the cyanylating reagent 1cyano-dimethylaminopyridiniumtetrafluoroborate (CDAP) is preferably used for the synthesis ofpolysaccharide-protein conjugates. The cyanilation reaction can beperformed under relatively mild conditions, which avoids hydrolysis ofthe alkaline sensitive polysaccharides. This synthesis allows directcoupling to a carrier protein.

The polysaccharide is solubilized in water or a saline solution. CDAP isdissolved in acetonitrile and added immediately to the polysaccharidesolution. The CDAP reacts with the hydroxyl groups of the polysaccharideto form a cyanate ester. After the activation step, the carrier proteinis added. Amino groups of lysine react with the activated polysaccharideto form an isourea covalent link. After the coupling reaction, a largeexcess of glycine is then added to quench residual activated functionalgroups. The product is then passed through a gel permeation column toremove unreacted carrier protein and residual reagents.

In another embodiment, the S. pneumonia conjugates may be combined withother polysaccharides, for example, N. meningitidis types A, C, W, Y, H.influenzae type B, S. aureus, S. epidermidis, Group B Streptococcus,Group A Streptococcus, etc. Preferably it is N. meningitidis (types Aand/or C are most preferred) and/or H. influenzae type B. Alternatively,the S. pneumonia conjugates of the invention may be combined with viralantigens, e.g., inactivated polio virus (IPV), influenza (inactivated,split subunit (e.g., F, G antigens)), etc. In another alternative, theS. pneumonia conjugates may be administered concomitantly with DTPa(diphtheria, tetanus, acellular pertussis) vaccines and DTPa combinationvaccines (DTPa±/Hepatitis B±IPV±H. influenzae type B). Preferred DTPavaccines have 25 Lf or less of Diphtheria toxoid. These additionalantigens may be in liquid form or lyophilized form.

In yet another embodiment, die present invention is an improved methodto elicit a (protective) immune response in infants (0-2 years old) byadministering a safe and effective amount of the vaccine of theinvention. Further embodiments of the present invention include theprovision of the antigenic S. pneumoniae conjugate compositions of theinvention for use in medicine and the use of the S. pneumoniaeconjugates of the invention in the manufacture of a medicament for theprevention (or treatment) of pneumococcal disease.

The present invention further provides an improved vaccine for theprevention or amelioration of pneumococcal infection in infants (e.g.,Otitis media), by relying on the addition of pneumococcal proteins to S.pneumoniae conjugate compositions of the invention. Preferably thepneumococcal protein is from the PhtX family (see below) to which may beadded further proteins. Such additional pneumococcal proteins maycomprise CbpX, CbpX truncates and Ply (see below), with the proviso thatthe selected S. pneumoniae surface proteins are different from the firstand secondary carrier proteins. One or more Moraxella catarrhalisprotein antigens can also be included in the combination vaccine. Thus,the present invention is an improved method to elicit a (protective)immune response against Otitis media in infants.

In yet another embodiment, the present invention is an improved methodto elicit a (protective) immune response in the elderly population (inthe context of the present invention a patient is considered elderly ifthey are 50 years or over in age, typically over 55 years and moregenerally over 60 years) by administering a safe and effective amount ofthe vaccine of the invention, preferably in conjunction with one, two,or possibly three S. pneumoniae surface proteins, with the proviso thatthe selected S. pneumoniae surface proteins are different from the firstand secondary carrier proteins. Preferably the pneumococcal protein isfrom the PhtX family (see below) to which may be added Ply andoptionally CbpX or CbpX truncates (see below).

The Streptococcus pneumoniae proteins of the invention are eithersurface exposed, at least during part of the life cycle of thepneumococcus, or are proteins which are secreted or released by thepneumococcus. Preferably the proteins of the invention are selected fromthe following categories, such as proteins having a Type II Signalsequence motif of LXXC (where X is any amino acid, e.g., thepolyhistidine triad family (PhtX)), choline binding proteins (CbpX),proteins having a Type I Signal sequence motif (e.g., Sp101), proteinshaving a LPXTG motif (where X is any amino acid, e.g., Sp128, Sp130),and toxins (e.g., Ply). Preferred examples within these categories (ormotifs) are the following proteins, or immunologically functionalequivalents thereof.

Preferably, the immunogenic composition of the invention comprises oneor more proteins selected from the group consisting of the PolyHistidine Triad family (PhtX), Choline Binding Protein family (CbpX),CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytX truncatechimeric proteins (or fusions), pneumolysin (Ply), PspA, PsaA, Sp128,Sp101, Sp130, Sp]25 and Sp133. However, if CbpX is PspC, then the secondprotein is not PspA or PsaA. More preferably, the immunogeniccomposition comprises 2 or more proteins selected from the groupconsisting of the Poly Histidine Triad family (PhtX), Choline BindingProtein family (CbpX), CbpX truncates, LytX family, LytX truncates, CbpXtruncate-LyCX truncate chimeric proteins (or fusions), pneumolysin(Ply), PspA, PsaA, and Sp128. More preferably still, the immunogeniccomposition comprises 2 or more proteins selected from the groupconsisting of the Poly Histidine Triad family (PhtX), Choline BindingProtein family (CbpX), CbpX truncates, and pneumolysin (Ply).

The Pht (Poly Histidine Triad) family comprises proteins PhtA, PhtB,PhtD, and PhtE. The family is characterized by a lipidation sequence,two domains separated by a proline-rich region and several histidinetriads, possibly involved in metal or nucleoside binding or enzymaticactivity, (3-5) coiled-coil regions, a conserved N-terminus and aheterogeneous C terminus. It is present in all strains of pneumococcitested. Homologous proteins have also been found in other Streptococciand Neisseria. Preferred members of the family comprise PhtA, PhtB andPhtD. More preferably, it comprises PhtA or PhtD. Most preferably itcomprises PhtD. It is understood, however, that the terms Pht A, B, D,and E refer to proteins having sequences disclosed in the citationsbelow as well as naturally-occurring (and man-made) variants thereofthat have a sequence homology that is at least 90% identical to thereferenced proteins. Preferably it is at least 95% identical and mostpreferably it is 97% identical.

With regards to the PhtX proteins, PhtA is disclosed in WO 98/18930, andis also referred to Sp36. As noted above, it is a protein from thepolyhistidine triad family and has the type II signal motif of LXXC.PhtD is disclosed in WO 00/37105, and is also referred to Sp036D. Asnoted above, it also is a protein from the polyhistidine triad familyand has the type II LXXC signal motif. PhtB is disclosed in WO 00/37105,and is also referred to Sp036B. Another member of the PhtB family is theC3-Degrading Polypeptide, as disclosed in WO 00/17370. This protein alsois from the polyhistidine triad family and has the type II LXXC signalmotif. A preferred immunologically functional equivalent is the proteinSp42 disclosed in WO 98/18930. A PhtB truncate (approximately 79 kD) isdisclosed in WO99/15675 which is also considered a member of the PhtXfamily. PhtE is disclosed in WO 00/30299 and is referred to as BVH-3.

Concerning the Choline Binding Protein family (CbpX), members of thatfamily were originally identified as pneumococcal proteins that could bepurified by choline-affinity chromatography. All of the choline-bindingproteins are non-covalently bound to phosphoryleholine moieties of cellwall teichoic acid and membrane-associated lipoteichoic acid.Structurally, they have several regions in common over the entirefamily, although the exact nature of the proteins (amino acid sequence,length, etc.) can vary. In general, choline binding proteins comprise anN terminal region (N), conserved repeat regions (R1 and/or R2), aproline rich region (P) and a conserved choline binding region (C), madeup of multiple repeats, that comprises approximately one half of theprotein. As used in this application, the term “Choline Binding Proteinfamily (CbpX)” is selected from the group consisting of Choline BindingProteins as identified in WO97/41151, PbcA, SpsA, PspC, CbpA, CbpD, andCbpG. CbpA is disclosed in WO97/41151. CbpD and CbpG are disclosed inWO0/29434. PspC is disclosed in WO97/09994. PbcA is disclosed inWO98/21337.SpsA is a Choline binding protein disclosed in WO 98/39450.Preferably the Choline Binding Proteins are selected from the groupconsisting of CbpA, PbcA, SpsA and PspC.

Another preferred embodiment is CbpX truncates wherein “CbpX” is definedabove and “truncates” refers to CbpX proteins lacking 50% or more of theCholine binding region (C). Preferably such proteins lack the entirecholine binding region. More preferably, such protein truncates lack (i)the choline binding region and (ii) retain the proline rich region andat least one repeat region (R1 or R2). More preferably still, thetruncate has 2 repeat regions (R1 and R2). Examples of such preferredembodiments are NR1×R2, NR1×R2P, R1×R2P and R1×R2 as illustrated inWO99/51266 or WO99/51188, however, other choline binding proteinslacking a similar choline binding region are also contemplated withinthe scope of this invention.

The LytX family is membrane associated proteins associated with celllysis. The N-terminal domain comprises choline binding domain(s),however the LytX family does not have all the features found in the CbpAfamily noted above and thus for the present invention, the LytX familyis considered distinct from the CbpX family. In contrast with the CbpXfamily, the C-terminal domain contains the catalytic domain of the LytXprotein family. The family comprises LytA, B and C. With regards to theLytX family, LytA is disclosed in Ronda et al., Eur J Biochem,164:621-624 (1987). LytB is disclosed in WO 98/18930, and is alsoreferred to as Sp46. LytC is also disclosed in WO 98/18930, and is alsoreferred to as Sp91. A preferred member of that family is LytC.

Another preferred embodiment are LytX truncates wherein “LytX” isdefined above and “truncates” refers to LytX proteins lacking 50% ormore of the Choline binding region. Preferably such proteins lack theentire choline binding region. Yet another preferred embodiment of thisinvention are CbpX truncate-LytX truncate chimeric proteins (orfusions). Preferably this comprises NR1xR2 (or R1xR2) of CbpX and theC-terminal portion (Cterm, i.e., lacking the choline binding domains) ofLytX (e.g., LytCCterm or Sp91Ctern). More preferably CbpX is selectedfrom the group consisting of CbpA, PbcA, SpsA and PspC. More preferablystill, it is CbpA. Preferably, LytX is LytC (also referred to as Sp91).Another embodiment of the present invention is a PspA or PsaA truncateslacking the choline binding domain (C) and expressed as a fusion proteinwith LytX. Preferably, LytX is LytC.

Pneumolysin is a multifunctional toxin with a distinct cytolytic(hemolytic) and complement activation activities (Rubins et al., Am .Respi. Cit Care Med, 153:1339-1346 (1996)). The toxin is not secreted bypneumococci, but it is released upon lysis of pneumococci under theinfluence of autolysin. Its effects include e.g., the stimulation of theproduction of inflammatory cytokines by human montocytes, the inhibitionof the beating of cilia on human respiratory epithelial, and thedecrease of bactericidal activity and migration of neutrophils. The mostobvious effect of pneumolysin is in the lysis of red blood cells, whichinvolves binding to cholesterol. Because it is a toxin, it needs to bedetoxified (i.e., non-toxic to a human when provided at a dosagesuitable for protection) before it can be administered in vivo.Expression and cloning of wild-type or native pneumolysin is known inthe art. See, for example, Walker et al. (Infect Immun, 55:1184-1189(1987)), Mitchell et al. (Biochim Biophys Acta, 1007:67-72 (1989) andMitchell et al (WAR, 18:4010 (1990)). Detoxification of ply can beconducted by chemical means, e.g., subject toGMBS, or formalin orglutarahdehye treatment or a combination of both. Such methods are wellknown in the art for various toxins. Alternatively, ply can begenetically detoxified. Thus, the invention encompasses derivatives ofpneumococcal proteins which may be, for example, mutated proteins. Theterm “mutated” is used herein to mean a molecule which has undergonedeletion, addition or substitution of one or more amino acids using wellknown techniques for site directed mutagenesis or any other conventionalmethod. For example, as described above, a mutant ply protein may bealtered so that it is biologically inactive whilst still maintaining itsimmunogenic epitopes, see, for example, WO90/06951, Berryetal. (InfectImmun, 67:981-985(1999)) and WO99/03884. As used herein, it isunderstood that the term “Ply” refers to mutated or detoxifiedpneumolysin suitable for medical use (i.e., non toxic).

With regards to PsaA and PspA, both are know in the art. For example,PsaA and transmembrane deletion variants thereof have been described byBerry & Paton, Infect Immun December 1996;64(12):5255-62. PspA andtransmembrane deletion variants thereof have been disclosed in, forexample, U.S. Pat. No. 5,804,193, WO 92/14488, and WO 99/53940.

Sp128 and Sp130 are disclosed in WO00/76540. Sp125 is an example of apneumococcal surface protein with the Cell Wall Anchored motif of LPXTG(where X is any amino acid). Any protein within this class ofpneumococcal surface protein with this motif has been found to be usefulwithin the context of this invention, and is therefore considered afurther protein of the invention. Sp125 itself is disclosed in WO98/18930, and is also known as ZmpB—a zinc metalloproteinase. Sp101 isdisclosed in WO 98/06734 (where it has the reference # y85993). It ischaracterized by a Type I signal sequence. Sp133 is disclosed in WO98/06734 (where it has the reference # y85992). It is also characterizedby a Type I signal sequence.

Examples of preferred Moraxella catarrhalis protein antigens which canbe included in a combination vaccine (especially for the prevention ofotitis media) are: OMP 106 [WO 97/41731 (Antex) & WO 96/34960 (PMC)];OMP21; LbpA &/or LbpB [WO 98/55606 (PMC)]; TbpA &/or TbpB [WO 97/13785 &WO 97/32980 (PMC)]; CopB [Helminen M E, et al. (1993) Infect. Immun.61:2003-201 0]; UspA1 and/or UspA2 [WO 93/03 761 (University of Texas)];OmpCD; HasR (PCT/EP99/03824); PilQ (PCT/EP99/03823); OMP85(PCT/EP00/01468); lipo06 (GB 9917977.2); lipo10 (GB 9918208.1); lipo11(GB 9918302.2); lipo18 (GB 9918038.2); P6 (PCT/EP99/03038); D15(PCT/EP99/03822); OmplA1 (PCT/EP99/0678 1); Hly3 (PCT/EP99/03257); andOmpE. Examples of non-typeable Haemophilus influenzae antigens which canbe included in a combination vaccine (especially for the prevention ofotitis media) include: Fimbrin protein [(U.S. Pat. No. 5,766,608—OhioState Research Foundation)] and fusions comprising peptides therefrom[eg LB1(f) peptide fusions; U.S. Pat. No. 5,843,464 (OSU) or WO99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281673 (StateUniversity of New York)]; TbpA and/or TbpB; Hia; Hsf; Hin47; Hif; Hmw1;Hmw2; Hmw3; Hmw4; Hap; D15 (WO 94/12641); P2; and PS (WO 94/26304).

As noted above, the proteins of the invention may also be beneficiallycombined. Preferred combinations include, but are not limited to,PhtD+NR1×R2, PhtD+NR1×R2P, PhtD+NR1×R2-Sp91Cterm chimeric or fusionproteins, PhtD+Ply, PhtD+Sp128, PhtD+PsaA, PhtD+PspA, PhtA+NR1×R2,PhtA+NR1×R2P, PhtA+NR1×R2-Sp91Cterm chimeric or fusion proteins,PhtA+Ply, PhtA+Sp128, PhtA+PsaA, PhtA+PspA, NR1×R2+LytC, NR1×R2P+PspA,NR1×R2+PspA, NR1×R2P+PsaA, NR1×R2+PsaA, NR¹×R2+Sp128, R1×R2+LytC,R¹×R2+PspA, R1×R2+PsaA, R1×R2+Sp128, R1×R2+PhtD, R1×R2+PhtA. Preferably,NR1×R2±P (or R1×R2±P) is from CbpA or PspC. More preferably it is fromCbpA. Other combinations include 3 protein combinations such asPhtD+NR1×R2P +Ply, PhtD+NR1×R2+Ply, PhtA+NR1×R2+Ply andPhtA+NR1×R2P+Ply.

The vaccines of the present invention are preferably adjuvanted.Suitable adjuvants include an aluminum salt such as aluminum hydroxidegel (alum) or aluminum phosphate, but may also be a salt of calcium,magnesium, iron or zinc, or may be an insoluble suspension of acylatedtyrosine, or acylated sugars, cationically or anionically derivatizedpolysaccharides, or polyphosphazenes. When adjuvanted with aluminumsalts, the ratio of aluminum salt to polysaccharide is less than 10:1(w/w). Preferably it is less than 8:1 and more than 2:1.

It is preferred that the adjuvant be selected to be a preferentialinducer of a TH1 type of response. Such high levels of Th1-typecytokines tend to favor the induction of cell mediated immune responsesto a given antigen, whilst high levels of Th2-type cytokines tend tofavor the induction of humoral immune responses to the antigen.

It is important to remember that the distinction of Th1 and Th2-typeimmune response is not absolute. In reality an individual will supportan immune response which is described as being predominantly Th1 orpredominantly Th2. However, it is often convenient to consider thefamilies of cytokines in terms of that described in murine CD4+ve T cellclones by Mosmann and Coffman (Mosmann, T. R. and Coffman, R L. (1989)TH1 and TH2 cells: different patterns of lymphokine secretion lead todifferent functional properties. Annual Review of Immunology, 7, p145-173). Traditionally, Th1-type responses are associated with theproduction of the INF-γ and IL-2 cytokines by T-lymphocytes. Othercytokines often directly associated with the induction of Th1-typeimmune responses are not produced by T-cells, such as IL-12. Incontrast, Th2-type responses are associated with the secretion of IL-4,IL-5, IL-6, IL-10. Suitable adjuvant systems which promote apredominantly Th1 response include: Monophosphoryl lipid A or aderivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A(3D-MPL) (for its preparation see GB 2220211 A); and a combination ofmonophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipidA, together with either an aluminum salt (for instance aluminumphosphate or aluminum hydroxide) or an oil-in-water emulsion. In suchcombinations, antigen and 3D-MPL are contained in the same particulatestructures, allowing for more efficient delivery of antigenic andimmunostimulatory signals. Studies have shown that 3D-MPL is able tofurther enhance the immunogenicity of an alum-adsorbed antigen [Thoelenet al. Vaccine (1998) 16:708-14; EP 689454-B1].

An enhanced system involves the combination of a monophosphoryl lipid Aand a saponin derivative, particularly the combination of QS21 and3D-MPL as disclosed in WO 94/00153, or a less reactogenic compositionwhere the QS21 is quenched with cholesterol as disclosed in WO 96/33739.A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210, andis a preferred formulation. Preferably the vaccine additionallycomprises a saponin, more preferably QS21. The formulation may alsocomprise an oil in water emulsion and tocopherol (WO 95/17210). Thepresent invention also provides a method for producing a vaccineformulation comprising mixing a protein of the present inventiontogether with a pharmaceutically acceptable excipient, such as 3D-MPL.Unmethylated CpG containing oligonucleotides (WO 96/02555) are alsopreferential inducers of a TH1 response and are suitable for use in thepresent invention.

The vaccine preparations of the present invention may be used to protector treat a mammal susceptible to infection, by means of administeringsaid vaccine via systemic or mucosal route. These administrations mayinclude injection via the intramuscular, intraperitoneal, intradermal orsubcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory, genitourinary tracts. Intranasaladministration of vaccines for the treatment of pneumonia or otitismedia is preferred (as nasopharyngeal carriage of pneumococci can bemore effectively prevented, thus attenuating infection at its earlieststage). Although the vaccine of the invention may be administered as asingle dose, components thereof may also be co-administered together atthe same time or at different times (for instance pneumococcalpolysaccharides could be administered separately at the same time or 1-2weeks after the administration of the bacterial protein component of thevaccine for optimal coordination of the immune responses with respect toeach other). For co-administration, the optional Th1 adjuvant may bepresent in any or all of the different administrations, however it ispreferred if it is present in combination with the bacterial proteincomponent of the vaccine. In addition to a single route ofadministration, 2 different routes of administration may be used. Forexample, polysaccharides may be administered IM (or ID) and bacterialproteins may be administered IN (or ID). In addition, the vaccines ofthe invention may be administered IM for priming doses and IM or IN(without aluminum) for booster doses.

The amount of conjugate antigen in each vaccine dose is selected as anamount which induces an immunoprotective response without significant,adverse side effects in typical vaccines. Such amount will varydepending upon which specific immunogen is employed and how it ispresented. Generally, it is expected that each dose will comprise0.1-100 μg of polysaccharide, for polysaccharide conjugates 0.1-50 μg ofpolysaccharide, preferably 1-10 μg, of which 1 to 5 μg is a preferredrange and 2-5 μg is a more preferable range. However, for serotype 6B,the preferred dosage will comprise 3-10 μg of polysaccharide, morepreferably 5-10 μg of polysaccharide conjugate.

The content of protein antigens in the vaccine will typically be in therange 1-100 μg, preferably 5-50 μg, most typically in the range 5-25 μg.Following an initial vaccination, subjects may receive one or severalbooster immunizations adequately spaced.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenuin Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

The vaccines of the present invention may be stored in solution orlyophilized. As a liquid, the vaccine of the invention is typicallystored in 0.5 ml solution/dose. Preferably the vaccine is adsorbed ontoan aluminum salt. If the solution is lyophilized, it is preferably inthe presence of a sugar such as sucrose or lactose or trelalose. It isstill further preferable that they are lyophilized and extemporaneouslyreconstituted prior to use. Lyophilizing of Streptococcuspolysaccharides may result in a more stable composition (vaccine) andmay possibly lead to higher antibody titers in the presence of 3D-MPLand in the absence of an aluminum based adjuvant.

Although the vaccines of the present invention may be administered byany route, administration of the described vaccines into the skin (ID)forms one embodiment of the present invention. Human skin comprises anouter “horny” cuticle, called the stratum corneum, which overlays theepidermis. Underneath this epidermis is a layer called the dermis, whichin turn overlays the subcutaneous tissue. Researchers have shown thatinjection of a vaccine into the skin, and in particular the dermis,stimulates an immune response, which may also be associated with anumber of additional advantages. Intradermal vaccination with thevaccines described herein forms a preferred feature of the presentinvention.

The conventional technique of intradermal injection, the “mantouxprocedure”, comprises steps of cleaning the skin, and then stretchingwith one hand, and with the bevel of a narrow gauge needle (26-31 gauge)facing upwards the needle is inserted at an angle of between 10-15°.Once the bevel of the needle is inserted, the barrel of the needle islowered and further advanced whilst providing a slight pressure toelevate it under the skin. The liquid is then injected very slowlythereby forming a bleb or bump on the skin surface, followed by slowwithdrawal of the needle.

More recently, devices that are specifically designed to administerliquid agents into or across the skin have been described, for examplethe devices described in WO 99/34850 and EP 1092444, also the jetinjection devices described for example in WO 01/13977; U.S. Pat. No.5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat. No. 5,334,144, US5,993,412, U.S. Pat. No. 5,649,912, U.S. Pat. No. 5,569,189, U.S. Pat.No. 5,704,911, U.S. Pat. No. 5,383,851, U.S. Pat. No. 5,893,397, U.S.Pat. No. 5,466,220, U.S. Pat. No. 5,339,163, U.S. Pat. No. 5,312,335,U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Pat. No.5,520,639, U.S. Pat. No. 4,596,556, U.S. Pat. No. 4,790,824, U.S. Pat.No. 4,941,880, U.S. Pat. No. 4,940,460, WO 97/37705 and WO 97/13537.Alternative methods of intradermal administration of the vaccinepreparations may include conventional syringes and needles, or devicesdesigned for ballistic delivery of solid vaccines (WO 99/27961), ortransdermal patches (WO 97/48440. WO 98/28037); or applied to thesurface of the skin (transdermal or transcutaneotus delivery WO98/20734; WO 98/28037).

When the vaccines of the present invention are to be administered to theskin, or more specifically into the dermis, the vaccine is in a lowliquid volume, particularly a volume of between about 0.05 ml and 0.2ml.

The content of antigens in the skin or intradermal vaccines of thepresent invention may be similar to conventional doses as found inintramuscular vaccines (see above). However, it is a feature of skin orintradermal vaccines that the formulations may be “low dose”.Accordingly the protein antigens in “low dose” vaccines are preferablypresent in as little as 0.1 to 10 μg, preferably 0.1 to 5 μg per dose;and the polysaccharide (preferably conjugated) antigens may be presentin the range of 0.01-1μg, and preferably between 0.01 to 0.5 μg ofpolysaccharide per dose.

As used herein, the term “intradermal delivery” means delivery of thevaccine to the region of the dermis in the skin. However, the vaccinewill not necessarily be located exclusively in the dermis. The dermis isthe layer in the skin located between about 1.0 and about 2.0 mm fromthe surface in human skin, but there is a certain amount of variationbetween individuals and in different parts of the body. In general, itcan be expected to reach the dermis by going 1.5 mm below the surface ofthe skin. The dermis is located between the stratum corneum and theepidermis at the surface and the subcutaneous layer below. Depending onthe mode of delivery, the vaccine may ultimately be located solely orprimarily within the dermis, or it may ultimately be distributed withinthe epidermis and the dermis.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLES Example 1

Determination of the Polysaccharides to which the Immune Response isRegulated with Age

Human antibody titers to both pre-immune and post-immunization (2 weeksto 3 months) polysaccharides (unconjugated) were collected eitherinternally or via external sources. FIG. 1 shows the relationshipbetween the immunogenicity of each serotype polysaccharide, as measuredby the geometric mean fold-increase in antibody titre (GFI) afterpolysaccharide immunization, and the mean age of the subjects in thestudy. The linear correlations of log geometric mean fold increase andage give an indication if the immune response is regulated with age. Asshown in FIG. 1, serotypes 6, 14, 19 and 23 are significantly correlatedwith age (p<0.001), whereas serotypes 8, 12 and 18 are lesssignificantly correlated with age (0.05<p<0.2). Finally, serotypes 1, 2,3, 4, 5, 7 and 9 are not significantly correlated with age (p>or =0.20).

Example 2

General Methodology of Determining Antibody Responses in Various Mammals

The sera were tested for IgG antibodies to the pneumococcalpolysaccharides by an ELISA based on a consensus assay for human seraproposed by the joint CDCAWHO workshops held between 1994 and 1996 (WHO1996, Plikatis et al J. Clin. Microbiol 38: 2043 (2000)). Briefly,purified capsular polysaccharides from ATCC (Rockville, Md., 20852) werecoated at 25 μg/ml in phosphate buffered saline (PBS) on high bindingmicrotitre plates (Nunc Maxisorp) overnight at 4 C. The plates wereblocked with 10% fetal calf serum (FCS), 1 hour at 37 C. Serum sampleswere pre-incubated with the 20 μg/ml cell-wall polysaccharide (StatensSerum Institute, Copenhagen) and 10% FCS at room temperature for 30minutes to neutralize antibodies to this antigen. A reference serum 89SF(courtesy of Dr. C Frasch, USFDA) was treated in the same fashion, andincluded on every plate. The samples were then diluted two-fold on themicroplate in 10% FCS in PBS, and equilibrated at room temperature for 1hour with agitation. After washing, the microplates were equilibratedwith peroxidase labelled anti-human IgG Fc monoclonal antibody(HP6043-HRP, Stratech Scientific Ltd) diluted 1:4000 in 10% FCS in PBSfor 1 hour at room temperature with agitation. The ELISA was performedto measure rat IgG using Jackson ImmunoLaboratories Inc.peroxidase-conjugated AffiniPure Goat anti-Rat IgG (H+L) (code112-035-003) at 1:5000. The titration curves were referenced to standardsera for each serotype using logistic log comparison by SoftMax Pro. Thepolysaccharide concentrations used to coat the ELISA plate have beenfixed at 10 μg/ml for all serotypes except 6B and 23F, where 20 μg/mlhas been used. In addition, 100% fetal calf serum was used as thediluent when testing antisera for serotype 6B, as this serotype wasprone to non-specific ELISA responses. Serology for serotype 3 on Rhesussera used mHSA comix for the coating antigen. Tile color was developedusing 4 mg OPD (Sigma) per 10 ml pH 4.5 0.1M citrate buffer with 14 μlH2O2 for 15 minutes in the dark at room temperature. The reaction wasstopped with 50 μl HCl, and the optical density was read at 490 nmrelative to 650 nm. IgG concentrations were determined by reference oftitration points to the calibration curve modeled using a 4-parameterlogistic log equation calculated by SoftMax Pro software.

To obtain absolute antibody concentrations in μg/ml, pooled referenceantisera were calibrated by two independent methods. For rat antisera,the method of Zollinger and Boslego (1981) was used for 11 serotypes,and for 4 serotypes this was compared with values obtained byimmunoprecipitation. Excellent correspondence was found between the twomethods. For murine sera, purified monoclonal IgGI antibodies were used,and their active concentrations were confirmed by corollary response(PVW 1999). In this case, reasonable correspondence was found. ForRhesus monkey sera, it was demonstrated that the anti-fgG reagents usedreact equally with human and Rhesus IgG; thus the calibrated humanreference sera 89SF (available from the US FDA) was employed toreference the ELISA.

The ELISA to measure the murine and rat IgG to the pneumococcalpolysaccharides was similar with the following exceptions. Locallymanufactured polysaccharides were used to coat the ELISA plates at 20μg/ml in PBS for serotypes 6B and 23F, and 10 μg/ml in PBS for serotypes14 and 19F. Jackson ImmunoLaboratories Inc. Peroxidase-conjugatedAffiniPure Goat Anti-mouse IgG (H+L) and AffiniPure Goat Anti-rat IgG(H+L) were employed to detect bound IgG. HP6043-HRP reacted equally withhuman and Rhesus purified IgG, and so this reagent was used for Rhesusantiserum, and the reference serum was using 89SF.

The reference serum for human and Rhesus serology was 89SF, kindlyprovided by Dr. Carl Frasch. The universally accepted weight-basedconcentration calibration values for the human reference serum 89SF for]gG, IgA and IgM against 10 pneumococcal serotypes using 2 differentmethods was published (Salazar et al).

The protein ELISA was performed similarly to the polysaccharide ELISAwith the following modifications. The protein was coated overnight at2.0 μg/ml in PBS. The serum samples were diluted in PBS containing 10%foetal calf serum and 0.1% polyvinyl alcohol. Bound human antibody wasdetected using Sigina Peroxydase-conjugated goat affinity purifiedantibody to Human IgG Fc (reference A-2290). To calibrate the proteinresponse in the human and Rhesus monkey serology, Sandoglobulin lot 069,found to contain significant anti-protein D antibody, was used as thereference and given an arbitrary value of 100 ELISA units. For murineand rat serology, the antibody concentrations were quantified byperforming corollary response by either direct antigen coating, or byantibody capture.

The sera were also tested for their ability to kill live pneumococci inan in vitro opsonophagocytic assay. The opsonophagocytosis assay wasadapted from the published protocol (Romero-Steiner et al. 1997), aswell as a detailed protocol provided by Sandy Steiner of the CDC as partof a multi-laboratory study.

Two methods were used. In method A, pneumococcal strains provided by theCDC were replaced by SB production strains were used. Secondly, theHL-60 cells were replaced by freshly purified human neutrophils (PMN).The results are expressed as the serum dilution required for 50%bacterial killing.

In method B, the CDC protocol was followed more closely from a publishedand detailed standardized protocol provided by the CDC as part of amulti-laboratory study (Romero-Steiner 1997, Romero-Steiner 2000).

Briefly, differentiated HL60 cells were centrifuged at 1000 rpm (300×g)and the culture supernatant was drawn off. The cells were resuspended inthe assay buffer consisting of HBSS-BSA. If antibiotics were present inthe culture media, this procedure was repeated to ensure completeremoval of antibiotics.

Serum samples were pre-diluted in advance for 4 assays to optimizevolume measurements. It was demonstrated that samples diluted 1:2 inassay buffer yielded stable opsonic titres for at least 5 days if keptat 4° C. Twenty-five μl of diluted sera was added to 25 μl of assaybuffer in a microplate round-bottom well. Twofold serial dilution wereperformed with 25 μl volume, again to optimize volume measurements.

Baby rabbit complement and pneumococcal cultures were kept at −70° C.until use. A 4:2:1 volume combination of activated HL60 cells, freshlythawed pneumococal culture and freshly thawed baby rabbit complement wasmixed with vortexing. Twenty-five μl of this mixture was rapidlydistributed to the microplate wells containing diluted sera, yielding afinal volume of 50 μl. This gave IE 5 HL60, 150 pneumococcal CFU and7.1% complement concentration per well in the final mixture, except forserotype 6B in which two modifications were made; the final complementconcentration was 12.5% and 5% FCS was included in the assay buffer toequalise growth of pneumococci during incubation. The microplates wereincubated for 2 hours at 37° C. with 5% CO2 with shaking at 210 rpm.

After incubation, a viable count was made of the pneumococci from a 20μl aliquot of the wells. Wells containing only assay buffer with noserum were used as the blank wells to determine the exact number ofpneumococci added per well. The mean number of CFU in 8 blank wells oneach plate was used for subsequent calculations.

The percent killing was calculated relative to the mean of the blankwells. The titre of a serum sample was determined by the maximumreciprocal dilution of serum able to facilitate greater than 50% killingof the pneulnococci. The values are reported as discontinuous titres of8, 16 32 etc. Samples for which there was less than 50% killing arereported with a titre <8. Samples in which a prozone effect was observedwere repeated, and the second result was taken. If a prozone effect wasobserved again, the result was considered invalid. This occurs in lessthan 5% of the samples. Samples which had a titre greater than 1024 wererepeated starting at a 1:64 dilution.

Example 3

Effects of Combination of Pneumococcal PS-PD Conjugates onImmunogenicity in Adult Rats

It has been observed that the combination of vaccines into multivalentformulations can result in the decrease in immunogenicity of one or morecomponents of the vaccine. This has been especially observed forconjugate vaccines, and has been called carrier-induced epitopicsuppression. The underlying mechanism for this suppression is not wellunderstood, but it tends to happen at higher dosages of carrier protein.

An 11-valent pneumococcal conjugate vaccine is an example of combinationvaccines. Since the combination of each serotype's conjugate will add tothe total amount of protein used to immunize, it is important todetermine whether the combination of each conjugate vaccine into amultivalent formulation results in a significant decrease in theimmunogenicity of the conjugate.

Protocol:

Adult rats were immunized with pneumococcal-polysaccharide protein Dconjugate vaccines (see, WO00/56360) either individually, or combined ina multivalent formulation. Groups of 10 rats were immunized twice 28days apart, and test bleeds were obtained on day 28 and day 42 (14 daysafter the second dose).

Antibody concentration was measured as described. The opsonic titreswere measured according to method A.

Results:

All conjugates induced specific IgG antibodies as measured by ELISA(FIG. 2). Opsonic activity (as determined by the reciprocal of thedilution of pooled sera able to kill 50% of live pneumococci) was alsodetected in all sera.

FIG. 2 also shows the effect of combination of monovalent PS-PDconjugates on their immunogenicity in adult rats, as measured by IgGconcentration and opsonic titre at 14 days post II.

Statistical analysis was performed on all samples to determine ifdifferences in IgG concentration upon combination were significant. Onlytype 14 showed a significant decrease in ELISA titers upon combination.The IgG concentration was reduced to levels that were similar to theother serotypes. All other differences were not significant, but type 7Fapproached significance (p=0.08).

Serotypes 1, 3, 6B, 9V and 23F actually show increases upon combination.

Example 4

Independent Variation of the Dosage of Serotypes 6B and 23F

Combination of individual conjugate vaccines into a multi-valentformulation results in increases or decreases of the antibody response.The immune regulation of the response is serotype dependent. Tocharacterize the immune response to a combined 11-Valent conjugatevaccine, an experiment was undertaken which combined the 11 valences intwo groups, 6B and 23F together, versus the remaining 9 valences.

Protocol:

Infant and Adult rats were immunized with 11-Valent PS-PD pneumococcalconjugate vaccine in a two-tiered dosage, that is, the 6B&23F dosagevaried independently from the other 9 valence, as shown in Table 1.TABLE 1 The 11-valent PS-PD Two-Way Dosage Formulation Dosage 1, 3, 4,5, Dosage 6B and 23F 7F, 9V, 14, 18C, Group (μg) 19F (μg) 1 0.01 0.01 20.01 0.1 3 0.01 1 4 0.1 0.01 5 0.1 0.1 6 0.1 1 7 1 0.01 8 1 0.1 9 1 1

Infant OFA rats were randomized to different mothers and were 7 days oldwhen they received the first immunization. Ten rats per group received 3immunizations on days 0, 14 and 28. Bleeds were performed on day 42 (14days post III) and 56 (28 days post III).

Results:

3D analysis of the two-tiered dosages indicates immune regulation ininfant rats caused by 6B-PD and 23F-PD. FIG. 3 shows the GMC for 11serotypes and PD versus the dosage of 6B and 23F in one dimension, andthe dosage of the 9 others in the second dimension. The trend is alwaysthe same for all serotypes and PD. Increasing the dosage of 6B and 23Fhas a dramatic effect on decreasing the antibody response to theremaining conjugates, even though the dosage of those conjugates isunchanged. This effect is very strong in the infant rats, but onlyslightly observable in the adult rats (not shown).

FIG. 4 shows the antibody concentration against each serotype in theconjugate vaccine as a function of total Protein D content. Ifcarrier-induced epitopic suppression was the principle or only cause ofreduction in the immune response with increasing vaccine dosage, it isexpected that these curves would be monotonically decreasing. Rather,the wave function indicates there is some other factor influencing theantibody response. As noted from FIG. 3, when the dosage is dividedcombining serotypes 6B and 23F, a smooth 3D surface is obtained,indicating that 6B and 23F regulate the immune response to the otherserotypes. Because in FIG. 4 serotype 6B does show a monotonicallydecreasing immune response, it may be surmised the dosage of serotype 6Bis the dominant factor, as its interaction with itself is alwaysconstant, and thus it only shows the effect of carrier-induced epitopicsuppression.

Conclusions:

Independent variation of the dosage of 6B&23F and the other 9 serotypesrevealed that the dosage of serotypes 6B&23F exerted an influence on theantibody response to the other serotypes. The antibody response to eachserotype was reduced with increasing total amount of PD immunized,indicating carrier-induced epitopic suppression, but since the relationis not smooth, there is an additional factor. In addition, the IgGresponse to PD also decreases with increasing dosage, opposite to whatis expected from carrier-induced epitopic suppression. Taken together,these imply a heretofore-unknown regulation of the immune response toconjugate vaccines mapped onto the dosage of serotypes 6B and 23F.

Example 5

Demonstration that the Immune Regulation from Serotypes 6B and 23F isTransmitted via the Protein Carrier.

Objective:

It is apparent that the dosage of conjugates 6B and 23F regulate theantibody response to the other conjugates in a multivalent formulation.The following experiment was performed to determine if the immuneregulation associated with 6B&23F-PD (conjugates) in infant rats was dueto the polysaccharide, or the polysaccharide protein conjugate.

Protocol:

Conjugates 6B&23F-PD or PS (unconjugated) were combined with otherserotypes in a multivalent formulation, with the dosage of 6B&23F at0.01 and 1.0 μg, and with the plain polysaccharide at 1.0 μg (without6B&23F conjugates).

Infant OFA rats were randomized to different mothers and were 7 days oldwhen they received the first immunization. Ten rats per group received 3immunizations on days 0, 14 and 28. Bleeds were performed on day 42 (14days post M).

Results:

As previously observed, an increase in the 6B&23F-PD dosage decreasedthe response to 19F. When PS replaced the conjugate, a higher responseto 19F was observed.

Conclusion:

The presence of a 1 μg dosage of 6B and 23F conjugate vaccine issufficient to regulate the immune response to serotype 19F in amultivalent conjugate vaccine, however, the same dosage of plainpolysaccharide has no effect. Since it has been determined thatserotypes 6B and 23F are regulated in their immune response in humansand animals, we may conclude the immune regulation of serotypes 6B and23F are transmitted to the other serotypes via the common proteincarrier.

Example 6

Modification of the Protein Carrier for Serotype 6B

The seroconversion rate of against 6B PS-conjugate was low in the infantrat at 0.1 ug dosage. Other factors that could influence theimmunogenicity of the conjugate were examined. These include the ratioof carbohydrate to protein present in the material, the specific methodof linkage used, the presence of free polysaccharide, and the specificcarrier protein used.

Modification of the coupling chemistry did not increase theimmunogenicity of the 6B conjugates in either the infant rat or mousemodels. It does appear that the use of the TT carrier increasesimmunogenicity in the mouse model, but only at a higher dosage.Conjugates were synthesized with an initial carrier protein (proteinD)/PS ration of 2.5:1. Other conjugates were synthesized with an initialcarrier protein (Protein D)/PS ratio of 1:1.

Example 7

Clinical Evaluations

Several vaccine formulations of the present invention are undergoingclinical evaluation in humans. Table 2 illustrates the composition ofsuch vaccines. TABLE 2 S. pneumoniae formulations Strep PS serotype:N^(o)1 N^(o)2 N^(o)3 N^(o)4 6B (μgPS -carrier) 10 μg -DT 5 μg -DT 10 μg-DT 5 μg -DT 19F (μgPS -carrier) 3 μg -DT 3 μg -DT 5 μg -PD 3 μg -PD 23F(μgPS -carrier) 5 μg -DT 5 μg -DT 5 μg -DT 5 μg -DT 1 (μgPS -carrier) 3μg -PD 3 μg -PD 5 μg -PD 3 μg -PD 3 (μgPS -carrier) 3 μg -PD 2 μg -PD 5μg -PD 3 μg -PD 4 (μgPS -carrier) 3 μg -PD 2 μg -PD 5 μg -PD 3 μg -PD 5(μgPS -carrier) 3 μg -PD 3 μg -PD 5 μg -PD 3 μg -PD 7F (μgPS -carrier) 3μg -PD 3 μg -PD 5 μg -PD 3 μg -PD 9V (μgPS -carrier) 3 μg -PD 2 μg -PD 5μg -PD 3 μg -PD 14 (μgPS -carrier) 3 μg -PD 2 μg -PD 5 μg -PD 3 μg -PD18C (μgPS -carrier) 3 μg -PD 2 μg -PD 5 μg -PD 3 μg -PD Total PScontent: 42 μg PS 32 μg PS 60 μg PS 37 μg PS Protein carrier content:˜22 μg PD ˜18 μg PD ˜42 μg PD ˜25 μg PD ˜18 μg DT ˜14 μg DT ˜15 μg DT˜10 μg DT Alum content (Al³⁺): ˜0.21 mg ˜0.16 mg ˜0.32 mg ˜0.19 mg +/−Combination with MenC_(ads lyo): μg PS 10 μg PSC 10 μg PSC μg TT ˜10 μgTT ˜10 μg TT μg Al³⁺ 0.05 mg Al³⁺ 0.05 mg Al³⁺

While the preferred embodiments of the invention are illustrated by theabove, it is to be understood that the invention is not limited to theprecise instructions herein disclosed and that the right to allmodifications coming within the scope of the following claims isreserved.

1. An improved Streptococcus pneumoniae vaccine comprising 11 or morepolysaccharides from different S. pneumonia serotypes conjugated to 2 ormore carrier proteins wherein, serotypes 6B, 19F and 23F are conjugatedto a first carrier protein and remaining serotypes are conjugated to 1or 2 secondary carrier proteins, and wherein the secondary carrierproteins are different from the first carrier protein.
 2. An improvedStreptococcus pneumoniae vaccine comprising 11 or more polysaccharidesfrom different S. pneumonia serotypes conjugated to 2 or more carrierproteins wherein, serotypes 6B, and 23 F are conjugated to a firstcarrier protein and remaining serotypes are conjugated to 1 or 2secondary carrier proteins, and wherein the secondary carrier proteinare different from the first carrier protein.
 3. An improvedStreptococcus pneumoniae vaccine comprising 11 or more polysaccharidesfrom different S. pneumonia serotypes conjugated to 2 or more carrierproteins wherein, serotype 6B is conjugated to a first carrier proteinand remaining serotypes are conjugated to 1 or 2 secondary carrierproteins, and wherein the secondary carrier protein are different fromthe first carrier protein.
 4. The vaccine of claim 1 wherein the firstcarrier protein is selected from the group consisting of DT, crm197, TT,Fragment C, Ply, PhtA, PhtB, PhtD, PhtE, OmpC and PorB.
 5. The vaccineof claim 1 wherein the secondary carrier protein comprises one or 2proteins selected from the group consisting of PD, DT, crm197, TT,Fragment C, Ply, PhtA, PhtB, PhtD, PhtE, OmpC and PorB.
 6. The vaccineof claim 1 wherein there is 1 secondary carrier protein.
 7. The vaccineof claim 1 wherein polysaccharides of each serotype are present in anamount of 1-10 μg.
 8. The vaccine of claim 7 wherein one or moreserotypes selected from the group consisting of 1, 3, 4, 5, 7F, 9V, 14and 18C are present in an amount of 2-5 μg.
 9. The vaccine of claim 1wherein the ratio of carrier protein to polysaccharide is 0.5 to 1.7(w/w).
 10. The vaccine of claim 9 wherein the ratio of carrier proteinto polysaccharide is from 0.7 to 1.5 for one or more serotypes selectedfrom the group consisting of 6B, 19F and 23F.
 11. The vaccine of claim 1wherein the secondary carrier protein is H. influenzae protein D (PD).12. The vaccine of claim 1 wherein polysaccharide serotype 6B isconjugated to a first carrier protein selected from the group consistingof DT, crm197 or TT.
 13. The vaccine of claim 12 wherein the firstcarrier protein is DT.
 14. The vaccine of claim 1 wherein polysaccharide6B is present in an amount of 5-10 μg/dose.
 15. The vaccine of claim 1further comprising unconjugated S. pneumoniae polysaccharides ofserotypes different from those conjugated, such that the number ofconjugated and unconjugated polysaccharides is less than or equal to 23.16. A method of eliciting a protective immune response to infants 0-2years old against S. pneumoniae by administering the vaccine of claim 1.17. A method of eliciting a protective immune response to the elderlyaged 50 years or over against S. pneumoniae by administering the (i)vaccine of claim 1 and (ii) a S. pneumoniae surface protein from thePhtX family, wherein the S. pneumoniae surface protein is different fromthe first and secondary carrier proteins.
 18. A method of eliciting aprotective immune response to infants 0-2 years old against Otitis mediaby administering the (i) vaccine of claim 1 and (ii) a S. pneumoniaesurface protein from the PhtX family, wherein the S. pneumoniae surfaceprotein is different from the first and secondary carrier proteins. 19.The method of claim 17 wherein the PhtX family protein is PhtD or PhtB.20. The method of claim 19 wherein the PhtX family protein is PhtD. 21.The method of claim 17 further comprising a CbpX family protein.
 22. Themethod of claim 21 wherein the CbpX protein is a truncate lacking thecholine binding domain.
 23. The method of claim 22 wherein the CbpXtruncate is choline binding protein A.
 24. The method of claim 17further comprising Ply.
 25. The vaccine of claim 2 wherein the firstcarrier protein is selected from the group consisting of DT, crm197, TT,Fragment C, Ply, PhtA, PhtB, PhtD, PhtE, OmpC and PorB.
 26. The vaccineof claim 2 wherein the secondary carrier protein comprises one or 2proteins selected from the group consisting of PD, DT, crm197, TT,Fragment C, Ply, PhtA, PhtB, PhtD, PhtE, OmpC and PorB.
 27. The vaccineof claim 2 wherein there is 1 secondary carrier protein.
 28. The vaccineof claim 2 wherein polysaccharides of each serotype are present in anamount of 1-10 μg.
 29. The vaccine of claim 28 wherein one or moreserotypes selected from the group consisting of 1, 3, 4, 5, 7F, 9V, 14and 18C are present in an amount of 2-5 μg.
 30. The vaccine of claim 2wherein the ratio of carrier protein to polysaccharide is 0.5 to 1.7(w/w).
 31. The vaccine of claim 30 wherein the ratio of carrier proteinto polysaccharide is from 0.7 to 1.5 for one or more serotypes selectedfrom the group consisting of 6B, 19F and 23F.
 32. The vaccine of claim 2wherein the secondary carrier protein is H. influenzae protein D (PD).33. The vaccine of claim 2 wherein polysaccharide serotype 6B isconjugated to a first carrier protein selected from the group consistingof DT, crm197 or TT.
 34. The vaccine of claim 33 wherein the firstcarrier protein is DT.
 35. The vaccine of claim 2 wherein polysaccharide6B is present in an amount of 5-10 μg/dose.
 36. The vaccine of claim 2further comprising unconjugated S. pneumoniae polysaccharides ofserotypes different from those conjugated, such that the number ofconjugated and unconjugated polysaccharides is less than or equal to 23.37. A method of eliciting a protective immune response to infants 0-2years old against S. pneumoniae by administering the vaccine of claim 2.38. A method of eliciting a protective immune response to the elderlyaged 50 years or over against S. pneumoniae by administering the (i)vaccine of claim 2 and (ii) a S. pneumoniae surface protein from thePhtX family, wherein the S. pneumoniae surface protein is different fromthe first and secondary carrier proteins.
 39. A method of eliciting aprotective immune response to infants 0-2 years old against Otitis mediaby administering the (i) vaccine of claim 2 and (ii) a S. pneumoniaesurface protein from the PhtX family, wherein the S. pneumoniae surfaceprotein is different from the first and secondary carrier proteins. 40.The method of claim 38 wherein the PhtX family protein is PhtD or PhtB.41. The method of claim 40 wherein the PhtX family protein is PhtD. 42.The method of claim 38 further comprising a CbpX family protein.
 43. Themethod of claim 42 wherein the CbpX protein is a truncate lacking thecholine binding domain.
 44. The method of claim 43 wherein the CbpXtruncate is choline binding protein A.
 45. The method of claim 38further comprising Ply.
 46. The vaccine of claim 3 wherein the firstcarrier protein is selected from the group consisting of DT, crm197, TT,Fragment C, Ply, PhtA, PhtB, PhtD, PhtE, OmpC and PorB.
 47. The vaccineof claim 3 wherein the secondary carrier protein comprises one or 2proteins selected from the group consisting of PD, DT, crm197, TT,Fragment C, Ply, PhtA, PhtB, PhtD, PhtE, OmpC and PorB.
 48. The vaccineof claim 3 wherein there is 1 secondary carrier protein.
 49. The vaccineof claim 3 wherein polysaccharides of each serotype are present in anamount of 1-10 μg.
 50. The vaccine of claim 49 wherein one or moreserotypes selected from the group consisting of 1, 3, 4, 5, 7F, 9V, 14and 18C are present in an amount of 2-5 μg.
 51. The vaccine of claim 3wherein the ratio of carrier protein to polysaccharide is 0.5 to 1.7(w/w).
 52. The vaccine of claim 51 wherein the ratio of carrier proteinto polysaccharide is from 0.7 to 1.5 for one or more serotypes selectedfrom the group consisting of 6B, 19F and 23F.
 53. The vaccine of claim 3wherein the secondary carrier protein is H. influenzae protein D (PD).54. The vaccine of claim 3 wherein polysaccharide serotype 6B isconjugated to a first carrier protein selected from the group consistingof DT, crm197 or TT.
 55. The vaccine of claim 54 wherein the firstcarrier protein is DT.
 56. The vaccine of claim 3 wherein polysaccharide6B is present in an amount of 5-10 μg/dose.
 57. The vaccine of claim 3further comprising unconjugated S. pneumoniae polysaccharides ofserotypes different from those conjugated, such that the number ofconjugated and unconjugated polysaccharides is less than or equal to 23.58. A method of eliciting a protective immune response to infants 0-2years old against S. pneumoniae by administering the vaccine of claim 3.59. A method of eliciting a protective immune response to the elderlyaged 50 years or over against S. pneumoniae by administering the (i)vaccine of claim 3 and (ii) a S. pneumoniae surface protein from thePhtX family, wherein the S. pneumoniae surface protein is different fromthe first and secondary carrier proteins.
 60. A method of eliciting aprotective immune response to infants 0-2 years old against Otitis mediaby administering the (i) vaccine of claim 3 and (ii) a S. pneumoniaesurface protein from the PhtX family, wherein the S. pneumoniae surfaceprotein is different from the first and secondary carrier proteins. 61.The method of claim 59 wherein the PhtX family protein is PhtD or PhtB.62. The method of claim 61 wherein the PhtX family protein is PhtD. 63.The method of claim 59 further comprising a CbpX family protein.
 64. Themethod of claim 63 wherein the CbpX protein is a truncate lacking thecholine binding domain.
 65. The method of claim 64 wherein the CbpXtruncate is choline binding protein A.
 66. The method of claim 59further comprising Ply.